Monitoring the compaction of single DNA molecules in Xenopus egg extract in real time

DNA compaction is required for the condensation and resolution of
chromosomes during mitosis, but the relative contribution of individual
chromatin factors to this process is poorly understood. We developed a
physiological, cell-free system using high-speed Xenopus egg extracts and
optical tweezers to investigate real-time mitotic chromatin fiber
formation and force-induced disassembly on single DNA molecules. Compared
to interphase extract, which compacted DNA by ~60%, metaphase extract
reduced DNA length by over 90%, reflecting differences in whole-chromosome
morphology under these two conditions. Depletion of the core histone
chaperone ASF1, which inhibits nucleosome assembly, decreased the final
degree of metaphase fiber compaction by 29%, while depletion of linker
histone H1 had a greater effect, reducing total compaction by 40%.
Compared to controls, both depletions reduced the rate of compaction, led
to more short periods of decompaction, and increased the speed of
force-induced fiber disassembly. In contrast, depletion of condensin from
metaphase extract strongly inhibited fiber assembly, resulting in
transient compaction events that were rapidly reversed under high force.
Altogether, these findings support a speculative model in which condensin
plays the predominant role in mitotic DNA compaction, while core and
linker histones act to reduce slippage during loop extrusion and modulate
the degree of DNA compaction.